Children born after assisted reproduction more commonly carry a mitochondrial genotype associating with low birthweight

Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.


Supplementary data to Figure 1 Supplementary Table 1. Prevalence of haplogroups per mode of conception.
Only the haplogroups that were present in more than 10 children were further considered in the analysis.Statistics were performed using a two-sided Fisher's exact test.ART N=270, SC N=181.% are calculated as the number of individuals with the haplogroup divided by the total number of individuals in the category ART or SC.ART: assisted reproductive technologies, SC: spontaneously conceived.

Table 2 . Prevalence of subhaplogroups per mode of conception.
Statistics were performed using a two-sided Fisher's exact test.ART N=270, SC N=181.% are calculated as the number of individuals with the subhaplogroup divided by the total number of individuals in the category ART or SC.ART: assisted reproductive technologies, SC: spontaneously conceived.

Table 3 . Distribution ART and SC individuals with homoplasmic variants outside of the haplogroup
. % are calculated as the number of individuals with a homoplasmic variant in the given region, divided by the total number of individuals in the category ART or SC.ART N=270, SC N=181.The sum of all the percentages does not add up to 100% because individuals can have multiple homoplasmic variants in the different categories.Synonymous and nonsynonymous variants are subcategories of protein-coding variants.Statistics were performed using a two-sided Fisher'

Supplementary Table 4. Distribution of the number of heteroplasmic variants per individual.
Supplementary Table 5. Location of the heteroplasmic variants.%are calculated as the number of individuals with a heteroplasmic variant in the given region, divided by the total number of individuals in the category ART or SC.ART N=270, SC N=181.Note that the total sum of the absolute numbers of each category does not add up to the total number of variants found in the ART and SC group because the insertions and deletions (ART: N=7 and SC: N=6) in the protein-coding regions are not subcategorized in the synonymous and non-synonymous categories.Statistics were performed using a two-sided Fisher's exact test.ART: assisted reproductive technologies, SC: spontaneously conceived.

Table 6 .
Type of heteroplasmic variants.% are calculated as the number of individuals with a heteroplasmic variant of a specific type, divided by the total number of individuals in the category ART or SC.ART N=270, SC N=181.Statistics were performed using a two-sided Fisher's exact test.ART: assisted reproductive technologies, SC: spontaneously conceived.

Table 7 . Mean sum of heteroplasmic loads of the individuals categorized according to their location.
Synonymous and non-synonymous variants are subcategories of protein-coding variants.Statistics were performed using a two-sided Mann-Whitney U Test. ART: assisted reproductive technologies, SC: spontaneously conceived.

Supplementary Table 10. Distribution of ART and SC individuals with homoplasmic variants outside of the haplogroup distributed whether their birthweight was categorized under or above the 10 th percentile
. % are calculated as the number of individuals with a certain homoplasmic variant, divided by the total number of individuals in the category <P10 (N=28) and >P10 (N=360).The percentages do not add up to 100% because individuals can have multiple homoplasmic variants in different categories.tRNA homoplasmies were more frequently found in the <P10 group, however, after correcting for multiple testing, this association was not statistically significant (p=0.04,p-values ≤0.007 were considered significant).Statistics were performed using a two-sided Fischer's exact test and corrected for multiple testing using the Bonferroni method.ART: assisted reproductive technologies, SC: spontaneously conceived.

Supplementary Table 11. Distribution of ART and SC individuals with homoplasmic variants outside of the haplogroup distributed whether their birthweight was categorized under or above the 25 th percentile
. % are calculated as the number of individuals with a certain homoplasmic variant, divided by the total number of individuals in the category <P25 (N=67) and >P25 (N=321).The percentages do not add up to 100% because individuals can have multiple homoplasmic variants in different categories.Statistics were performed using a two-sided Fischer's exact test.ART: assisted reproductive technologies, SC: spontaneously conceived.

Supplementary Table 12. Distribution of SC individuals with heteroplasmic variants distributed whether their birthweight was categorized under or above the 10 th percentile
. % are calculated as the number of individuals with a certain heteroplasmic variant, divided by the total number of individuals in the category <P10 (N=9) and >P10 (N=155).The percentages do not add up to 100% because individuals can have multiple heteroplasmic variants in different categories.Statistics were performed using the two-sided Fischer's exact test.SC: spontaneously conceived.

Table 26. Changes in heteroplasmic load after transmission seen in the oocytes of natural and OS cycles from the same .
No differences were seen between natural and stimulated oocytes, Chi Square, p=0.265